The present invention relates to a method for forecasting and evaluating the pharmacokinetic parameter of an injection containing lipid A analog, a quality evaluating method of the injection for ensuring the injection to have constant pharmacokinetic parameter, and a preparation process of the injection.
Lipid A is the main moiety causing activities of lipopolysaccharide (hereinafter referred to as LPS), has various biological activities such as macrophage stimulation, antitumor effect and pyrogenicity (for example, Haruhiko Takada and Shozo Kotani, Protein, Nucleic Acid and Enzyme, 31(4), 361 (1986)).
Various lipid A analogs have recently been synthesized and examined for their biological activities (Yuji Ogawa et al., Metabolism 26(5), 415 (1989)). Most of the Lipid A analogs having the glycolipid structure are sparingly soluble in water, so that it is difficult to prepare an injection with lipid A analogs.
In order to prepare an injection and to obtain a highly transparent aqueous solution, the addition of triethylamine, bovine serum albumin, lipids, or the like (Y. B. Kim, et al, Eur. J. Biochem. 31, 230 (1972) and R. B. Ramsey, et al, Blood, 56, 307 (1980), J. Dijkstra, et al, J. Immunol., 138, 2663 (1987)) and use of basic amino acids or polyamines (JP-A 4-198192) as a solubilizing agent have been reported.
On the other hand, as a method of dispersing a lipid such as lecithin or the like in water to form aggregates of liposomes or the like, known is a method to add a lipid to a buffer having a pH around neutrality, followed by heating and sonication.
The inventors have prepared an injection preparation having a high transparency, which contains aggregates having a diameter not greater than 30 nm, prepared by dissolving the lipid A analog or a pharmacologically acceptable salt thereof produced according to the methods disclosed in JP-A 5-194470 and WO96/39411 in an alkaline aqueous solution and then adding a buffer thereto.
Administration of this injection preparation to the living body of a rat or a beagle is however accompanied with the problem that the blood level of a lipid A analog shows a large variation, depending on the difference in the raw material medicament or preparation lot. This occurs because the state of the aggregates of the medicament (lipid) in the solution of the injection preparation is not uniform.
It is the common practice to evaluate the state of the aggregates of a lipid in a solution from the appearance observed through an electron microscope, particle size distribution measured by a laser diffraction particle size distribution measuring apparatus or consideration based on the physical properties such as critical micelle concentration or surface tension.
Not so many reports have however been proposed on circular dichroism spectroscopy and/or membrane fluidity evaluation method employed as an evaluation method of the invention.
In circular dichroism spectroscopy (which will hereinafter be abbreviated as xe2x80x9cCD spectroscopyxe2x80x9d), measurement is carried out reflecting the difference in the refractive index and absorbance of an optically active substance between those relative to left hand circular polarized light and those relative to right hand circular polarized light. This method is frequently employed for the conformation analysis of peptides or proteins or optical activity analysis of low-molecular compounds, but it has hardly been employed for the analysis of lipid-related substances. Reported are only the case wherein a dynamic change of a lipid in a liposome membrane depending on temperature was investigated by the analysis of variations in a CD spectrum (JP-A 62-252795); the case wherein CD spectroscopy is useful for evaluation of the characteristics of lipid particles in water, is an evaluation method markedly simple in operation and permits measurement of a dynamic state change in the membrane at a diluted concentration (N. Nakashima et al, CHEM. LET., 1503, 10 (1985)); and a case wherein CD spectroscopy is employed as one of the evaluation methods of the correlation between the characteristics of aggregates in various formulations of Prostaglandin E1 in liposome and elution characteristics (Sharon M. K. et al, Biochim. Biophys. Acta, 1327, 97 (1997)).
As a method for evaluating membrane fluidity (softness of membrane), known are fluorescence probe method, electron spin resonance (ESR) method and nuclear magnetic resonance (NMR) method (Robert B Gennith; Biomembrane, p146 (1992), published by Springer Verlag, Tokyo). Among them, the fluorescence probe method is a method for evaluating membrane fluidity having a bimolecular membrane structure of phospholipid. In this method, the state of the membrane in the vicinity of a fluorescent substance is observed by mixing a fluorescence probe such as diphenylhexatriene (which will hereinafter be abbreviated as xe2x80x9cDPHxe2x80x9d) in the membrane of lipid and then, measuring the polarity of fluorescence emitted upon exposure to polarized incident light. There is a report of the application of this method to a lipid A analog (Braudenburg K, et al., Biochim. Biophys. Acta, 225, 775 (1984)). The fluorescence probe method makes it possible to calculate fluorescence polarity (P: ranging from 0 to 0.5) and/or fluorescence anisotropy (r: ranging from 0 to 0.4) and/or order parameter (S: ranging from 0 to 1.0) by measuring the vertical polarizing component and horizontal polarizing component of fluorescence emitted from a lipid sample (Hiroshi Terada, Tetsuro Yoshimura; Liposome in Life Science (1992), published by Springer Verlag, Tokyo). Herein, as the order parameter (S) approaches 0, the membrane fluidity becomes larger. As it approaches 1, on the other hand, the membrane fluidity becomes smaller.
No report has however been published yet on the definite forecasting or evaluation of the pharmacokinetic parameter of a lipid A analog based on the correlation between the measuring and evaluating results of the aggregate condition of the medicament in a solution and the pharmacokinetic parameter. In addition, a report has been made neither on a process for producing an injection preparation wherein the pharmacokinetic parameter of a lipid A analog has been controlled nor a quality assurance method for ensuring the injection preparation to have predetermined pharmacokinetic parameter, each from the viewpoints of the state of the aggregates of the medicament (lipid) in a solution.
It is possible to prepare an injection preparation containing aggregates having a diameter not greater than 30 nm by dissolving a lipid A analog or pharmacologically acceptable salt thereof in an aqueous alkaline solution and then adding a buffer thereto. In the resulting injection preparation, the lipid A analog or pharmacologically acceptable salt thereof constitutes endoplasmic reticulum with lipid biomolecular membrane or micelle structure. In other words, an injection of a lipid A analog which has high transparency in the form of an aqueous solution, a preferred pH range as an injection and good stability can be prepared by the above-described process.
The injection preparation thus prepared is however accompanied with the problem that when it is administered to a rat or beagle, the blood level varies largely from one lot to another of a raw material medicament or the preparation. This owes to that the existing state of a lipid A analog in a solution, that is, the aggregate structure in the form of endoplasmic reticulum of lipid biomolecular membrane or micelle having a diameter not greater than 30 nm differs with the lot of the raw material medicament or injection preparation. There is accordingly a strong demand for the development of a practically usable injection of a lipid A analog, that is, an injection having uniform pharmacokinetic parameter, which is typified by the blood level, without being influenced by the difference among the lots of a raw material medicament or injection preparation; and a forecasting and evaluating method of the pharmacokinetic parameter of the injection.
With the foregoing in view, the present inventors have carried out an extensive investigation to search for an injection containing a lipid A analog, having high transparency and having good stability and in addition, exhibiting constant uniform pharmacokinetic parameter free from the influence of variations of a raw material medicament or injection preparation among lots; and forecasting and evaluating method of the pharmacokinetic parameter of the injection. As a result, it has been found that the object can be attained by the below-described constitutions, leading to the completion of the present invention.
In one aspect of the present invention, there is provided a forecasting method of the pharmacokinetic parameter of a lipid A analog in an injection preparation containing the lipid A analog or pharmacologically acceptable salt thereof, which comprises measuring membrane fluidity and/or circular dichroism in a solution.
In another aspect of the invention, there is also provided an evaluation method of the pharmacokinetic parameter of a lipid A analog in an injection preparation containing the lipid A analog or a pharmacologically acceptable salt thereof, which comprises measuring membrane fluidity and/or circular dichroism in a solution.
In a further aspect of the present invention, there is also provided a quality assurance method of an injection preparation containing a lipid A analog or a pharmacologically acceptable salt thereof, which comprises measuring and evaluating membrane fluidity and/or circular dichroism in a solution, and ensuring the lipid A analog to exhibit constant pharmacokinetic parameter in vivo.
The forecasting method of the pharmacokinetic parameter according to the present invention can be employed for the evaluation of an injection preparation, quality evaluation for obtaining an injection preparation exhibiting constant pharmacokinetic parameter or preparation of the injection preparation.
According to the present invention, an injection preparation which is transparent and is ensured to have stable pharmacokinetic parameter is available using a lipid A analog or a pharmacologically acceptable salt (which will hereinafter be called xe2x80x9clipid A analogxe2x80x9d, collectively). This is an object of the present invention. In addition, the present invention provides a forecasting and evaluating method of the pharmacokinetic parameter of an injection preparation containing the lipid A analog by measuring the membrane fluidity and/or circular dichroism; and also provides a quality evaluating method of an injection preparation containing a lipid A analog, which ensures the lipid A analog to exhibit constant pharmacokinetic parameter. This is another object of the present invention.
The lipid A analog of the present invention is represented by the following chemical structure, and can be produced by the process disclosed in, for example, JP-A 5-194470 or WO96/39411. 
wherein at least one of R1, R2, R3 and R4 is 
wherein each L is O, N or C; each M is O or N; each E independently is an integer of 0 to 14; each G independently is N, O, S, SO or SO2; each m independently is an integer of 0 to 14; each n independently is an integer of 0 to 14; each p independently is an integer of 0 to 10; each q independently is an integer of 0 to 10, the rest of R1, R2, R3 and R4 are, independently of one another, 
wherein each L is O, N or C; each M is O or N; each x independently is an integer of 0 to 14; each y independently is an integer of 0 to 14; each z independently is an integer of 0 to 14; each G independently is N, O, S, SO or SO2,
A1 and A2 are, independently of one another, H, OH, OCH3, 
wherein each d independently is an integer of 0 to 5; each f independently is an integer of 0 to 5; each g independently is an integer of 0 to 5; each A3 independently is 
wherein each j independently is an integer of 0 to 14, X is H, (CH2)tCH3, (CH2)tOH, (CH2)tO(CH2)vCH3, (CH2)tOPO(OH)2, (CH2)txe2x80x94CHxe2x95x90CHxe2x80x94(CH2)vCH3, (CH2)txe2x80x94Oxe2x80x94R5, 
wherein t and v, are independently of one another, an integer of 0 to 14; R5 is any of the above definitions of R1 to R4,
Y is H, OH, O(CH2)wCH3, a halogen atom, 
wherein w is an integer of 0 to 14. 
wherein R1 is a group selected from the groups consisting of 
wherein J, K and Q are each a linear or branched alkyl group of 1 to 15 carbon atoms; L is O, NH2 or CH2; M is O or NH; G is NH, O, S, SO or SO2,
R2 is a linear or branched alkyl group of 5 to 15 carbon atoms, R3 is a group selected from the groups consisting of 
wherein E is N, O, S, SO or SO2; A, B and D are each a linear or branched alkyl group of 1 to 15 carbon atoms,
R4 is a group selected from the groups consisting of a linear or branched alkyl group of 4 to 20 carbon atoms and 
wherein U and V are each a linear or branched alkyl group of 2 to 15 carbon atoms; W is a hydrogen atom or a linear or branched alkyl group of 1 to 5 carbon atoms,
R5 is a group selected from the groups consisting of a hydrogen atom, Jxe2x80x2, xe2x80x94Jxe2x80x2xe2x80x94OH, xe2x80x94Jxe2x80x2xe2x80x94Oxe2x80x94Kxe2x80x2, xe2x80x94Jxe2x80x2xe2x80x94Oxe2x80x94Kxe2x80x2xe2x80x94OH and xe2x80x94Jxe2x80x2xe2x80x94Oxe2x80x94PO(OH)2, wherein Jxe2x80x2 and Kxe2x80x2 are each a linear or branched alkyl group of 1 to 5 carbon atoms,
R6 is a group selected from the groups consisting of a hydroxyl group, a halogen atom, an alkoxy group of 1 to 5 carbon atoms, and an acyloxy group of 1 to 5 carbon atoms, A1 and A2 are, independently of one another, a group selected from the groups consisting of 
wherein Z is a linear or branched alkyl group of 1 to 10 carbon atoms.
Preferred examples of the lipid A analog for use in the present invention include 6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-enoyloxydecyl]-2-[3-oxo-tetradecanoylamido]-b-O-phosphono-a-D-glucopyranose]tetrasodium, a-D-glucopyranose, 3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-(3-methoxydecyl)-6-O-methyl-2-[(1-oxo-11-octadecenyl)amino]-4-O-phosphono-b-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-, 1-(dihydrogenphosphate), disodium[6(2Z, 3R)]; and a-D-glucopyranose, 3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-(3-methoxydecyl)-6-O-methyl-2-[(1-oxo-11-octadecenyl)amino]-4-O-phosphono-b-D-glucopyranosyl]-2-((1,3-dioxotetradecyl)amino]-, 1-(dihydrogenphosphate), tetrasodium[6(2Z, 3R)]. These compounds are represented by the following chemical structural formulae (III) and (IV). 
It is known that the blood level of liposome of phospholipid is influenced largely when it is introduced into a net system such as liver via a scavenger receptor. As a result of study on the mechanism of a scavenger receptor which recognizes and scavenges an anionic high molecule such as liposome, it is also known that membrane fluidity (softness of membrane), surface charge and particle diameter play important roles in the recognition of the scavenger receptor (Hiroshi Terada, Tetsuro Yoshimura; Liposome in Life Science, p326 (1992), published by Springer Verlag, Tokyo).
No example of evaluating physicochemical properties of the membrane of a preparation containing a lipid A analog has so far been proposed from the viewpoint of pharmacokinetic control.
The present invention has revealed that in a step for preparing an injection preparation which contains aggregates having a diameter not greater than 30 nm by dissolving a lipid A analog in an alkaline solution and then adding a buffer to the resulting solution, a pharmacokinetic profile of the injection containing a lipid-A analog can be controlled by measuring and evaluating its membrane fluidity and circular dichroism.
The membrane fluidity of the particles in an injection preparation using DPH has a close correlation with a blood level profile. Upon administration of the injection preparation, a preparation having larger membrane fluidity (a preparation having softer membrane) disappears more slowly from the blood larger AUC), while a preparation having smaller membrane fluidity (a preparation having harder membrane) disappears more rapidly from the blood (smaller AUC). This is because after administration of a lipid-A-containing injection, membrane fluidity affects the pharmacokinetic parameter of the medicament. The larger the membrane fluidity, the lipid A does not tend to be trapped easily by a scavenger receptor and is therefore not taken in a phagocyte such as liver cells readily, retarding the disappearance of lipid A from the circulating blood. The present invention has also revealed that there is a correlation between the CD spectrum change and variations in the pharmacokinetic parameter.
The present invention makes it possible to prepare an injection preparation containing a lipid-A analog ensured to have good pharmacokinetic parameter by utilizing, for the measurement of membrane fluidity and/or circular dichroism, the fact that membrane fluidity and/or circular dichroism in the solution of the injection differs definitely between a formulation of a low disappearing rate and a formulation of a high disappearing rate from the blood. The present invention also makes it possible to provide a forecasting or evaluating method of the pharmacokinetic parameter of an injection preparation containing a lipid-A analog by measuring membrane fluidity and/or circular dichroism; and a quality evaluating method for ensuring the injection to have constant pharmacokinetic parameter.
In the present invention, a highly-transparent injection preparation which has controlled pharmacokinetic parameter and contains aggregates having a diameter not greater than 30 nm can be prepared by dissolving a lipid A analog in an aqueous alkaline solution, adding thereto a buffer and measuring membrane fluidity and/or circular dichroism in the resulting solution. In the present invention, the membrane fluidity of an injection preparation is measured by the fluorescence probe method. This is a method for evaluating membrane fluidity of a bimolecular membrane structure of a phospholipid. Described specifically, the state of the membrane in the vicinity of a fluorescent substance is observed by mixing a fluorescence probe in the membrane of a lipid and measuring the polarity of fluorescence emitted upon exposure to polarized incident light. In the present invention, any one or more than one parameter selected from fluorescence polarity (P: ranging from 0 to 0.5), fluorescence anisotropy (r: ranging from 0 to 0.4) and order parameter (S: ranging from 0 to 1.0) may be used for evaluation of membrane fluidity. As a fluorescence probe to be used, any one capable of emitting stable fluorescence can be employed. Examples include diphenylhexatriene (DPH), carboxyfluorescein, calcein, Nile Red, pyrene and perylene.
For example, as the order parameter (S) approaches 0, the membrane fluidity is larger, while it approaches 1.0, the membrane fluidity is smaller. The order parameter (S) of the lipid A analog represented by the formula (III) or (IV) is usually 0.5 to 0.7, preferably 0.1 to 0.6, more preferably 0.1 to 0.5.
The circular dichroism spectroscopy (CD spectroscopy) of an injection preparation employed in the present invention is useful as a method for controlling or forecasting the pharmacokinetic parameter of a lipid A analog. It is preferred to evaluate the CD spectrum of each of a number of injection preparations and select the wavelength at which a large difference in CD intensity can be recognized. In the CD spectroscopy of the lipid A analog represented by the formula (III) or (IV), wavelength measured is usually 260 to 320 mm, preferably 270 to 310 mm, more preferably 280 to 300 mm.
As the alkaline aqueous solution in the present invention, the hydroxide of an alkali metal such as sodium hydroxide and potassium hydroxide, preferably sodium hydroxide may be used. The concentration thereof generally ranges from 0.0001M to 0.1M, preferably from 0.0005M to 0.01M and more preferably from 0.001 M to 0.01 M.
In the present invention, after an alkaline aqueous solution is added to a lipid A analog, the temperature of the mixture may be elevated. The elevated temperature must be higher than the phase transition temperature of the lipid A analog or a pharmacologically acceptable salt thereof, but has no more limitation. The elevated temperature generally ranges from 30xc2x0 C. to 60xc2x0 C., preferably 45xc2x0 C. to 55xc2x0 C. The stirring time generally ranges from 10 minutes to 3 hours. Stirring may be carried out with a conventional apparatus. When a lipid A analog is dissolved in a heated alkaline aqueous solution, a lipid A analog may be added to an alkaline aqueous solution which is heated in advance. Alternatively, after a lipid A analog is added to an alkaline aqueous solution, the mixture may be heated. In the present invention, the purpose of elevating the temperature is to accelerate hydration of lipid A analogs to improve dispersibility by elevating the temperature to the phase transition temperature of lipid A analogs or higher, to thereby obtain a transparent solution by stirring for a shortened period of time.
The lipid A analog represented by the formula (IV) however may be heated or stirred at room temperature for 10 minutes to 1.5 hours.
Examples of the component of the buffer for use in the present invention include phosphates, Tris(hydroxymethyl)aminomethane, citrates, glycine and the like. The concentration of the buffer generally ranges from 1 mM to 20 mM. The final pH value of the aqueous solution of the lipid A analog is preferably 4 to 9, more preferably 6 to 8, still more preferably 6.8 to 7.8. The final pH value may be adjusted by adding a solution of sodium hydroxide, hydrochloric acid, or the like after the addition of the buffer.
If necessary, addition of saccharides and/or amino acids to the buffer may give a more preferable result. In this case, saccharides and/or amino acids to be added may be either one, or two or more kinds thereof. Examples of the saccharide include milk sugar (lactose), sorbitol, glucose, trehalose, mannitol, dextran, and the like. Examples of the amino acid include neutral amino acids such as glycine, acidic amino acids such as aspartic acid, and basic amino acids such as arginine.
In the present invention, the resulting lipid A analog aqueous solution may be freeze-dried by a conventional method to obtain a freeze-dried preparation. Namely, lipid A or an analog thereof is dissolved in an alkaline aqueous solution, further stirred at an elevated temperature if necessary, followed by addition of a buffer to adjust pH of the mixture. After sterilizing filtration, the mixture is filled into a vial or the like, followed by freeze-drying to give a freeze-dried preparation.
When a preparation for injection according to the present invention is administered in the form of aqueous solution, the osmotic pressure ratio of the preparation is preferably adjusted to a value suitable for administrating to humans, generally around 1.
One example of each of the measuring methods of membrane fluidity and circular dichroism (CD spectroscopy) of an injection preparation and the evaluation method of the pharmacokinetic parameter in a rat administered with the preparation will next be described.
It will be evaluated using as a parameter fluorescence polarity (P) and/or fluorescence anisotropy (r) and/or order parameter (S) in accordance with the fluorescence probe method.
First, a solution having a concentration of 0.5 to 0.6 mg/ml is prepared as an injection preparation containing a lipid-A analog (a freeze-dried preparation in a 1.5 to 1.6 mg/vial is prepared by adding 3 ml of distilled water). Then, 4 xcexcl of a fluorescence probe solution obtained by dissolving 1.5 mg of DPH in 10 ml of THF is added, followed by sufficient mixing. The resulting mixture is allowed to stand at 50xc2x0 C. for 1 hour. After cooling to room temperature, P and/or r of the reaction mixture is measured by a fluorescent spectrophotometer. The measuring conditions of a fluorescent spectrophotometer xe2x80x9cModel F-450xe2x80x9d (trade name; product of Hitachi Ltd.) are as follows: excited wavelength of 360 nm, luminous wavelength of 428 mm, applied voltage of 700V, measuring temperature of 25xc2x0 C. and slit width of 5 nm. The order parameter (S) is calculated based on the equation: S=(r/0.398)1/2.
It can be judged that membrane fluidity is larger (the membrane is softer) at S closer to 0 and it is smaller (the membrane is harder) at S closer to 1. Measuring method of circular dichroism (CD spectroscopy) of the preparation
A solution having a concentration of 0.5 to 0.6 mg/ml is prepared as an injection containing a lipid A analog (a freeze-dried preparation in a 1.5 to 1.6 mg/vial is prepared by adding 3 ml of distilled water). The CD intensity of this preparation is evaluated by xe2x80x9cSpectropolarimeter J720WIxe2x80x9d (trade name; product of JASCO Corporation). Measurement is conducted under the conditions of wavelength of 200 to 500 nm (280 nm when fixed wavelength is employed), measuring temperature at room temperature, cell length of 1 cm, scanning speed of 20 nm/min and integration of 5 times.
Evaluation method of pharmacokinetic parameter in a rat administered with an injection preparation
The pharmacokinetic parameter profile of the preparation is judged using as a parameter an area under the curve available from the time-dependent curve of the blood level of medicament (this area will hereinafter be abbreviated as xe2x80x9cAUCxe2x80x9d).
First, as an injection preparation containing a lipid A analog, a solution having a concentration of 0.6 mg/ml is prepared by adding distilled water for injection. The resulting solution is administered (0.6 mg/Kg) to a male SD rat in a dose of 1 ml/Kg from its femoral vein. In addition to the blood collection prior to administration, the blood is collected 2, 5, 15, 30, 60 and 120 minutes after administration, each in an amount of 0.25 ml. Each of the bloods thus collected is centrifuged, whereby the plasma is obtained. From the plasma, the lipid A analog is extracted by liquid-liquid extraction, followed by labeling with fluorescence by using an ADAM reagent (product of Funakoshi Co., Ltd.). After solid layer extraction, the concentration of the lipid A analog contained in the plasma is measured using high performance liquid chromatography in accordance with the fluorescence detection method. The AUC is calculated from a change in the blood level from 0 to 120 minutes.
According to the present invention, it is possible to provide an injection preparation containing a lipid A analog as that having high transparency and stably controlled pharmacokinetic parameter. Also a forecasting method and evaluating method of the pharmacokinetic parameter of an injection preparation and a quality assurance method of an injection preparation containing a lipid A analog, which ensures the preparation to have constant pharmacokinetic parameter can be provided. The following are the advantage examples of the present invention.